Structural and functional roles of a conserved proline residue in the α2 helix of Escherichia coli thioredoxin
Structural and functional roles of a conserved proline residue in the α2 helix of Escherichia coli thioredoxin
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DOI：
10.1093/protein/10.12.1425
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发表时间：
1997-12-01
期刊：
影响因子：
--
通讯作者：
Decottignies, P
中科院分区：
文献类型：
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作者：
de Lamotte-Guéry, F;Pruvost, C;Decottignies, P
大肠杆菌硫氧还蛋白中的脯氨酸40位于α 2螺旋内的氧化还原活性位点（Cys32-Cys35）附近。该残基在大多数硫氧还毒素中的保存表明，它可能在该蛋白的结构和/或功能中起重要作用。我们利用定点诱变技术用Pro40代替Ala，在大肠杆菌中表达了突变体P40A。通过分子动力学模拟分析和比较了突变对硫氧还蛋白生物物理和生物学特性的影响。模型预测，Ala取代Pro40引起活性位点的位移，使Trp31暴露于溶剂中，并在螺旋α 2和α 3之间打开一个裂缝。溶剂化自由能（SFE）计算也表明，随着W31的易接近性，P40A的疏水性增强。这些预测与实验结果完全一致。突变体P40A表现出与野生型（WT）蛋白截然不同的色谱行为和荧光特性，这与W31的位移有关。P40A展开自由能的测定表明，突变体在2.9 kcal/mol的作用下发生全局失稳。然而，突变对转变曲线的影响非常不寻常，因为展开转变的中点增加了，这表明一些局部结构实际上被突变稳定了。尽管有这些结构上的改变，蛋白质还原叶绿体酶的能力和它与细菌还原酶的反应性都没有下降。唯一的功能差异是P40A在空气条件下对nadp -苹果酸脱氢酶的光激活中具有更高的稳定性，这表明突变体的再氧化速度低于WT。因此，可以得出结论，Pro40不是维持硫氧还蛋白氧化还原功能所必需的，而是蛋白质稳定性所必需的。
Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha 2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha 2 and alpha 3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.